Fundamental to understanding brain function is the ability to relate the spatio-temporal firing patterns of specific neurons to their functional connectivity and determine the strength and experience-dependent regulation of those connections. Genetically-encoded optical indicators have revolutionized both endeavors, enabling action potentials and synaptic transmission to be detected, but these approaches face two major hurdles: 1) overly dense expression often makes it impossible to trace the morphology and hence connectivity of specific neurons, and 2) there has been no method to measure the probability of synaptic transmission (Pr) and quantal size of synapses in vivo, leading synaptic strength connections and the mechanism of experience- dependent change unresolved. The first problem emerges from a lack of ability to target activity indicators to specific cells that are few enough in number so that their morphology and physical connectivity could be determined in a densely packed environment, to then permit the physical picture to be related to activity and connectivity. To meet this challenge, we propose a generalizable strategy for the creation of turn-on genetically-encoded activity indicators. These indicators are rationally modified from some of the best existing indicators of neural activity and synaptic transmission. The re-engineering enables the indicators to be activated by light to provide a Golgi-like view of cell morphology and report on action potentials and synaptic input. Because the indicators are turned on by light (unlike in the random labeling methods like the Golgi stain), one can select specific target cells and functionally image densely packed cells whose processes heavily overlap while knowing which process belongs to which cell, thereby permitting a simple form of elegant connectivity mapping. The second problem emerges from the lack of a method for synapse-specific quantal analysis. Large-scale quantal resolution imaging of synaptic responses would represent a powerful addition to the experimental neuroscience toolkit to help address how dynamic changes in synaptic strength contribute to sensation, action, learning and memory. Despite a wealth of knowledge on synaptic function in reduced ex vivo preparations, such as brain slices, due to the lack of effective tools, our knowledge of synaptic function in vivo during learning and behavior is extremely limited. A new approach that overcomes this technical gap would bridge the divide between synaptic and circuit level analyses in awake, behaving animals. High signal-to-noise spine level calcium imaging in behaving animals could address this gap. To meet this challenge we propose to develop synaptically-targeted calcium indicators that enable excitatory synaptic transmission to be imaged with quantal resolution simultaneously at hundreds to thousands of connections. Because this is an imaging method, it provides synapse-specific information that one cannot readily obtain from electrophysiological recordings that lump together measurements from a large number of inputs distributed over a neuron's dendritic tree. Optical quantal analysis in behaving animals would permit direct assessment of the dynamic fluctuations in synaptic efficacy that may underlie learning. It will also open whole new avenues of research that could explore how changes in synaptic efficacy contribute to fundamental aspects of sensation, action, and higher cognitive function. The collaboration between Isacoff, Scott and Adesnik enables these new tools to be validated for in vivo applications in brain circuit analysis and behavior in three model organisms: zebrafish, fruitfly and mouse.